[unreadable]-cell apoptosis contributes to loss of [unreadable]-cells and decreases in [unreadable]-cell function in both types 1 and 2 diabetes mellitus. It is therefore important to understand the mechanisms underlying [unreadable]-cell apoptosis if this process is to be prevented or delayed. Our hypothesis in the 1st project period (10/04-7/09) was that the group VIA Ca2+-independent phospholipase A2 (iPLA2[unreadable]) participates in ER stress-induced [unreadable]-cell apoptosis. We observed that (a) ER stress promotes accumulation and activity of iPLA2[unreadable] in the ER and mitochondria, (b) iPLA2[unreadable] activation during ER stress increases ceramide generation via neutral sphingomyelinase (NSMase)-catalyzed hydrolysis of sphingomyelins triggering the mitochondrial apoptotic pathway and [unreadable]-cell apoptosis, (c) these outcomes are suppressed by inhibition of iPLA2[unreadable] or NSMase, (d) iPLA2[unreadable]-null islets are less and iPLA2[unreadable]-transgenic (Tg) islets more sensitive to ER stress, (e) NSMase expression is unaffected in iPLA2[unreadable]-null islets and amplified in iPLA2[unreadable]-Tg islets, and (f) [unreadable]-cell iPLA2[unreadable] and NSMase messages are higher in the Akita mouse model of spontaneous [unreadable]-cell ER stress that leads to diabetes. We also find that iPLA2[unreadable] participates in [unreadable]-cell apoptosis induced by hyperglycemia and cytokines and that islet iPLA2[unreadable] and NSMase messages are elevated in pre-diabetic NOD mice. We hypothesize that iPLA2[unreadable]-mediated ceramide generation and triggering of mitochondrial abnormalities are critical contributory events to [unreadable]-cell apoptosis. The studies proposed herein are designed to elucidate the precise role of iPLA2[unreadable] in this process and whether this pathway, activated by ER stress, is also elicited by hyperglycemia and cytokines. Our Aims will address the following: Aim 1, role of iPLA2[unreadable] activation and iPLA2[unreadable]-derived lipid mediators. Hyperglycemia and cytokines promote [unreadable]-cell apoptosis, in part, by inducing ER stress and the role of iPLA2[unreadable] and generation of iPLA2[unreadable]-derived lipid mediators in this process will be assessed;Aim 2, induction of iPLA2[unreadable] and ceramide-generating pathway. SREBP-1 and CaMKII[unreadable] are known to affect iPLA2[unreadable] and are activated during ER stress. Their roles in iPLA2[unreadable] induction and the requirement of iPLA2[unreadable] activity for ceramide generation will be examined;Aim 3, requirement of iPLA2[unreadable] mobilization and activation to trigger mitochondrial abnormalities. The affects of genetic modulation, mobilization, and organelle-specific expression of iPLA2[unreadable] on the mitochondrial apoptotic pathway will be examined;Aim 4, sensitivity to ER stress following in vivo modulation of iPLA2[unreadable]. A role of iPLA2[unreadable] in modulating ER stress in [unreadable]-cells will be tested by crossing iPLA2[unreadable]-null with ER stress-sensitive Akita mice and iPLA2[unreadable]-Tg with ER stress-resistant CHOP-null mice;and Aim 5, contribution of iPLA2[unreadable] to [unreadable]-cell apoptosis during the evolution of autoimmune DM. The dependence of cytokine-induced [unreadable]-cell apoptosis on an iPLA2[unreadable]/nitric oxide (NO)-dependent pathway and the contribution of iPLA2[unreadable] to [unreadable]-cell apoptosis in NOD mice will be assessed. These Aims will be addressed using apoptosis, flow cytometry, immunoblotting, qRT-PCR, enzymatic activity assay, confocal microscopy, molecular biology, and mass spectrometry protocols. Findings from our studies will lead to increased understanding of iPLA2[unreadable] biology in the [unreadable]-cell. The long-range goal of our laboratory is to assess the role of iPLA2[unreadable] in [unreadable]-cell apoptosis during the development and progression of diabetes mellitus. PUBLIC HEALTH RELEVANCE: [unreadable]-cell apoptosis contributes to decreases in [unreadable]-cell mass and [unreadable]-cell dysfunction during the evolution of diabetes mellitus and is therefore important to understand the mechanisms underlying [unreadable]-cell apoptosis if this process is to be prevented or delayed. Observations in the PI's laboratory indicate involvement of iPLA2[unreadable] in the [unreadable]-cell apoptotic pathway and we propose to examine in greater detail the role of iPLA2[unreadable], as it relates to its expression, activation, and localization, and of iPLA2[unreadable]-derived lipid mediators in [unreadable]-cell apoptosis due to stimuli that contribute to [unreadable]-cell death during the evolution of diabetes mellitus. Findings from these studies will extend our knowledge of factors that adversely affect [unreadable]-cell health and identify targets for future therapeutic interventions to prevent [unreadable]-cell death.